Voltage sensitive dyes (VSD) are used to record and image spatial and temporal patterns of electrical activity in cells and tissues. This laboratory ha been engaged in the design, synthesis and application of VSDs. The dyes that have emerged from this effort are widely used to optically record electrical signals where electrode-based measurements would be unsuitable. In this proposed continuation of our research program, we plan to develop better dyes and use them to elucidate a fundamental issue in synaptic signaling. We also will engage in a variety of collaborative projects that will span scales from the subcellular to the clinic in both neuronal and cardiac systems. The first Aim proposes to build on the success of the new fluorinated VSDs with their improved photostability and sensitivity. We will modify these chromophores to adapt them to experiments that require different membrane binding, solubility and spectral properties to serve both our own experiments and those of our collaborators. Example collaborations include: marrying optical recording with optogenetic stimulation in hippocampal slices from transgenic mice; use of optical parametric oscillators for 2-photon excitation of long wavelength VSDs in brain slices and in vivo; merging optical recording with STED microscopy to image voltage in spines at nanometer resolution; imaging action potential propagation within single cardiomyocytes via rapid laser positioning; in vivo imaging of electrical activity in the heart; characterization of action potential propagation aroun lesions in human hearts. The second Aim proposes to develop a completely novel mechanism for voltage sensing - dipper dyes. As opposed to existing mechanisms which may produce at most a 60%/100mV change in fluorescence, the dipper dyes can in principle produce a several fold response to an action potential. In the third Aim, we will apply VSDs in combination with non-linear optical microscopy to investigate the voltage change at individual dendritic spines in response to neurotransmitter. These experiments, to be performed in mouse brain slices using 2-photon microscopy, will directly address the question of whether the spine can serve as an electrical compartment. We will also explore how electrical signals from pairs of neighboring spines can be integrated in the dendrite.